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The level of monocyte turnover predicts disease progression in the macaque model of AIDS

Atsuhiko Hasegawa, Huining Liu, Binhua Ling, Juan T. Borda, Xavier Alvarez, Chie Sugimoto, Heather Vinet-Oliphant, Woong-Ki Kim, Kenneth C. Williams, Ruy M. Ribeiro, Andrew A. Lackner, Ronald S. Veazey and Marcelo J. Kuroda

Data supplements

Article Figures & Data

Figures

  • Figure 1

    In vivo BrdU labeling of bone marrow cells in a rhesus macaque. (A) Bone marrow cells obtained from 6 distant bones (right and left humeri, femurs, and tibias) 24 hours after a single dose (60 mg/kg) of BrdU. The cells were stained for the presence of BrdU and Ki-67. Ki-67 was used to better distinguish BrdU+ cells. Numbers in the top right quadrants indicate the percentages of BrdU+ Ki67 bright bone marrow cells. (B) In vivo BrdU labeling does not affect total white blood cell counts. The total white blood cell (WBC) number in blood was monitored after BrdU injection in 7 uninfected (○) and 9 SIV/SHIV-infected (●) rhesus monkeys. Data show means ± SDs of each group per time point. Statistical analysis was performed by a repeated measure ANOVA. Data at each time point were also compared with data at time point 0 by a Dunnett test.

  • Figure 2

    Percentage of BrdU-labeled monocytes at 24 hours after BrdU administration as a reliable marker of monocyte turnover. (A) Kinetics of BrdU-labeled monocytes of 4 rhesus macaques after BrdU injection. Blood monocytes were defined as CD14+ cells after gating through CD45 and side scatter plots. Any T lymphocytes, B cells, and NK cells were further excluded by gating on CD3CD8CD20HLA-DR+CD45+ population. Data shown is the percentage of BrdU+ monocytes at indicated time points after BrdU injection. Note the inverse relation in the rank order of the percentage of BrdU labeling at 1 day versus 6 days. (B) Percentage of BrdU-labeled monocytes observed at 24 hours after BrdU administration inversely correlates with the time those cells spend in circulation. In other words, animals with the greatest percentage of CD14+BrdU+ cells at 1 day have the lowest percentage of CD14+ cells by 6 days after labeling. Data show the kinetics of BrdU-labeled monocytes from animal CA50 (red) and N195 (green) that exhibit a high and low percentage of labeled monocytes 24 hours after BrdU administration, respectively. The dotted line highlights the differences in the dynamic behavior of the 2 representative animals within 24 hours of BrdU injection that correlate with the decline of the BrdU+ monocytes in circulation.

  • Figure 3

    Increased monocyte turnover in SIV/SHIV-infected rhesus macaques. (A) Histograms of BrdU-labeled blood monocytes in uninfected and SIV/SHIV-infected animals and in animals with AIDS at 24 hours after BrdU treatment. Representative data from 2 animals per group are shown. Percentage values in histograms indicate BrdU+ CD14+ monocytes. (B) The percentage of BrdU-labeled monocytes in uninfected (n = 17) and chronically SIV- or SHIV-infected monkeys (n = 24) and in monkeys with AIDS (n = 5). P values were calculated by the Mann-Whitney U test. More information on the infected monkeys used in this study is provided in Table 1. Horizontal bars indicate the average percentage of BrdU+ CD14+ monocytes in each group. (C) Blood monocyte counts in monkeys before (n = 17; total 155 time points) and after infection (n = 17; total 156 time points) and in other monkeys with AIDS (n = 5; total 5 time points). Each bar indicates the average of monocyte counts in an individual monkey. P values were calculated by the Wilcoxon matched pairs test for the comparison between before and after infection and by the Mann-Whitney U test for the comparison between before infection and AIDS group. Error bars indicate SD of monocyte counts.

  • Figure 4

    Increased macrophages turnover in mesenteric lymph node from SIV-infected (AIDS) macaque. Lymph nodes were collected 24 hours after BrdU injection from uninfected and SIV-infected macaques. The percentages of BrdU+ monocytes in the uninfected and infected monkeys shown in this figure were 3.0% and 45.3%, respectively. Group data are shown in Table 3. Triple-label confocal microscopy was performed for CD163 (macrophage marker, blue), BrdU (red) to identify recently arrived macrophages, and TUNEL (green) to identify apoptotic macrophages. (A,B) From follicular areas and (C,D) from the cortical sinus areas in mesenteric lymph nodes from an uninfected animal (A,C) and an SIV-infected animal (B,D). Each panel is representative of 5 different fields of the follicular and the cortical sinus areas. Confocal microscopy was performed using a Leica TCS SP2 confocal microscope equipped with 3 lasers (Leica Microsystems). An oil objective 40× fluotar/NA 1.0 was used to image the slides. Scale bar, 74 μm. Magnification, × 40.

  • Figure 5

    High monocyte turnover predicts AIDS progression in SIV-infected rhesus macaques. (A) Kinetics of plasma viral load (left) in 4 SIV-infected rhesus macaques at multiple time points after SIV infection. The percentages of BrdU-labeled monocytes (middle) and CD3+ T lymphocytes (right) at indicated time points from the same 4 SIV-infected animals are shown. BrdU staining of peripheral blood was performed at 24 hours after BrdU injection at indicated time points. †Death of the animal. In parentheses is indicated the date the animal died after the last BrdU injection experiment (182 days after infection). (B) Correlation of BrdU+CD8+ T cells with other BrdU-labeled cells in infected monkeys. Note that the turnover of monocytes is not correlated with that of other cell types examined. (C) Relation between the percentage of BrdU-labeled cells and plasma viral loads in chronically SIV- or SHIV-infected rhesus macaques. Peripheral bloods were obtained from 21 infected macaques at 24 hours after BrdU inoculation and stained for BrdU. Dots represent individual animals. The Spearman rank test was used to determine the correlation between the percentage of BrdU+ cells and viral loads. (D) Correlation between the survival time after the in vivo BrdU experiment and the percentage of BrdU+ cells, CD4 counts, and plasma viral load at the time of the BrdU injection experiment. The only parameter that predicted progression to AIDS with statistical significance was high monocyte turnover. The Spearman rank correlation test was used to determine correlations and P values. The regression line and the 95% confidence intervals are shown with continuous and broken lines, respectively (applied only for panel B).

  • Figure 6

    No correlation between monocyte turnover and blood CD4 T-cell count in SIV- or SHIV-infected rhesus macaques. Peripheral blood samples were obtained from 21 chronically infected rhesus macaques at 24 hours after BrdU inoculation, and the percentage of BrdU-labeled monocytes and blood CD4 count were measured. The Spearman rank test was used to determine the correlation between the percentage of BrdU+CD14+ monocytes and CD4+ T-cell counts.

Tables

  • Table 1

    SIV- or SHIV-infected rhesus macaques used in this study

    Animal no.Virus straindpi* when inoculated with BrdUdpi* when euthanizedCause of death
    CN77SIVmac2517879AIDS
    BE86SIVmac251826827AIDS
    EL95SIVmac2515455AIDS
    EB54SIVmac251497498AIDS
    BH25SIVmac25110161017AIDS
    BT51SIVmac2518121044AIDS
    DN85SIVmac2518990Non-AIDS
    DR55SIVmac2519091Non-AIDS
    EB31SIVmac2518990Non-AIDS
    ED13SIVmac2518990Non-AIDS
    EB17SIVmac251111112Non-AIDS
    CE45SIVmac239182512AIDS
    FA97SIVmac239182316AIDS
    FB04SIVmac239182904§Still alive
    V754SIVmac239182792AIDS
    CT83SIVmac2397981062Non-AIDS
    DD19SIVmac239385811Non-AIDS
    DR58SIVmac239385558Non-AIDS
    EC40SIVmac239385597Non-AIDS
    EN51SIVmac239167552Non-AIDS
    EN84SIVmac239385569AIDS
    N708SIV B67011911192Non-AIDS
    BE63SHIV Ku1135387AIDS
    BV98SHIV Ku11351198§Still alive
    CT81SHIV Ku1119122Non-AIDS
    CR80SHIV 162p4135407Non-AIDS
    CA50SHIV 162p4149407AIDS
    CL85SHIV 162p4119122Non-AIDS
    • * dpi indicates days postinfection.

    • AIDS was defined when AIDS was diagnosed by pathology at necropsy. Non-AIDS was defined when no classic sign of AIDS was detected by pathology at necropsy.

    • Animals used for Figure 5D.

    • § Animals still alive at indicated date.

  • Table 2

    Calculated export/emigration rates of monocytes from bone marrow, s, and loss rates, δ for the 4 rhesus macaques

    Calculated rate parameters
    Animal no. (% BrdU+ monocytes at 24 h)Parameter
    sδ
    CA50 (38.1)6.7900.163
    BT51 (21.6)5.6920.135
    R842 (9.89)4.3250.111
    N195 (2.67)4.1490.093
  • Table 3

    High macrophage turnover in mesenteric lymph node of SIV-infected (AIDS) monkey

    Animal/areaTotalCD163+ cells (macrophage)
    BrdU+TUNEL+TotalBrdU+TUNEL+TUNEL+BrdU+
    Uninfected (3.0%)*
        Sinus73.8 ± 26.66.6 ± 2.340.8 ± 2.92.0 ± 1.60.4 ± 0.50
        Follicle90.8 ± 25.17.4 ± 3.937.0 ± 18.42.0 ± 1.40.4 ± 0.50.2 ± 0.4
    Infected (45.3%)
        Sinus189.0 ± 25.127.6 ± 7.8194.6 ± 11.033.6 ± 3.511.0 ± 1.42.0 ± 1.0
        Follicle169.2 ± 87.533.8 ± 11.9162.4 ± 43.227.0 ± 7.010.2 ± 1.81.0 ± 0.7
    P
        Sinus.0001.0004< .0001< .0001< .0001na§
        Follicle.0904.0015.0003< .0001< .0001.0650
    • * Percentages of BrdU+ monocytes in blood are indicated.

    • Data were analyzed for statistical significance by the unpaired t test.

    • Each value represents mean ± SD of the cell number per area obtained from 5 randomly selected areas.

    • § na indicates not applicable.

    • Not significant (P > .05).